SV40 DNA replication can be divided into four stages: 1) initiation of DNA synthesis, 2) propagation of the replication fork, 3) segregation of the newly synthesized progeny molecules, 4) maturation of the segregated progeny DNA by a gap filling process. These different stages of SV40 DNA replication are being studied with the aid of in vitro nuclei systems. The SV40 A gene product is required for initiation of DNA synthesis and assays are being developed to look for gene A product function in vitro. Both crude extracts and purifed preparations of SV40 tumor antigen (purified by immunological assay) are being employed. Cellular functions like untwisting activty, RNase, H, DNA polymerase activities, etc. are being purified and assayed in the in vitro system. Gap circle intermediates of SV40 DNA are being tested for the presence of ribonucleotides when they are produced in the presence or absence of inhibitors like hydroxyurea and cycloheximide. The gap has been localized at the terminus of replication in SV40 DNA.